Reproduction of selected plant varieties by tissue culture has been a commercial success for many years. The technique has enabled mass production of genetically identical selected ornamental plants, agricultural plants and forest species. The woody plants in this last group have perhaps posed the greatest challenges. Some success with conifers was achieved in the 1970s using organogenesis techniques wherein a bud, or other organ, was placed on a culture medium where it was ultimately replicated many times. The newly generated buds were placed on a different medium that induced root development. From there, the buds having stems and roots were planted in soil.
While conifer organogenesis was a breakthrough, costs were high due to the large amount of handling needed. There was also some concern about possible genetic modification. It was a decade later before somatic embryogenesis achieved a sufficient success rate so as to become the predominant approach to conifer tissue culture. With somatic embryogenesis, an explant, usually a seed or seed embryo, is placed on an initiation medium where it multiplies into a multitude of genetically identical immature embryos. These can be held in culture for long periods and multiplied to bulk up a particularly desirable clone. Ultimately, the immature embryos are placed on a development medium where they grow into somatic analogs of mature seed embryos. As used in the present description, a “somatic” embryo is a plant embryo developed by the laboratory culturing of totipotent plant cells or by induced cleavage polyembryogeny, as opposed to a zygotic embryo, which is a plant embryo removed from a seed of the corresponding plant. These embryos are then individually selected and placed on a germination medium for further development. Alternatively, the embryos may be used in artificial seeds, known as manufactured seeds.
There is now a large body of general technical literature and a growing body of patent literature on embryogenesis of plants. Examples of procedures for conifer tissue culture are found in U.S. Pat. Nos. 5,036,007 and 5,236,841 to Gupta et al.; U.S. Pat. No. 5,183,757 to Roberts; U.S. Pat. No. 5,464,769 to Attree et al.; and U.S. Pat. No. 5,563,061 to Gupta. Further, some examples of manufactured seeds can be found in U.S. Pat. No. 5,701,699 to Carlson et al., the disclosure of which is hereby expressly incorporated by reference. Briefly, a typical manufactured seed is formed of a seed coat (or a capsule) fabricated from a variety of materials such as cellulosic materials, filled with a synthetic gametophyte (a germination medium), in which an embryo surrounded by a tube-like restraint is received. After the manufactured seed is planted in the soil, the embryo inside the seed coat develops roots and eventually sheds the restraint along with the seed coat during germination.
One of the more labor intensive and subjective steps in the embryogenesis procedure is the selective harvesting from the development medium of individual embryos suitable for germination (e.g., suitable for incorporation into manufactured seeds). The embryos may be present in a number of stages of maturity and development. Those that are most likely to successfully germinate into normal plants are preferentially selected using a number of visually evaluated screening criteria. A skilled technician evaluates the morphological features of each embryo embedded in the development medium, such as the embryo's size, shape (e.g., axial symmetry), cotyledon development, surface texture, color, and others, and manually plucks desirable embryos out of the development medium with a pair of tweezers. The plucked desirable embryos are then carefully laid out on a tray in a two-dimensional array for further processing. This is a highly skilled yet tedious job that is time consuming and expensive. Further, it poses a major production bottleneck when the ultimate desired output will be in the millions of plants.
It has been proposed to use some form of instrumental image analysis for embryo selection to supplement or replace the visual evaluation described above. For example, PCT Application Ser. No. PCT/US00/40720 (WO 01/13702 A2) discloses an embryo delivery system for manufactured seeds including an imaging camera, which acquires and digitally stores images of embryos. The images are then sent to a computer, which classifies the embryos according to their desirability (i.e., likelihood to germinate and grow into normal at plants) based on predetermined parameters (axial symmetry, cotyledon development, surface texture, color, etc.) using a classification method disclosed in PCT Application Ser. No. PCT/US99/12128 (WO 99/63057). Those embryos that are classified as desirable are thereafter removed by mini-robotic pick and place systems and inserted into manufactured seeds. The disclosure of these two PCT applications is hereby expressly incorporated by reference.
While instrumental imaging analysis and subsequent automatic insertion of desirable embryos into manufactured seeds have been successful in increasing the efficiency of the embryogenesis procedure, there has not been a complete automated process of harvesting embryos, e.g., removing embryos from a development medium, sorting embryos according to their size/shape and singulating them into discrete units (e.g., by removing any undesirable tissues or other debris), and classifying them according to their desirability for incorporation into manufactured seeds. In other words, there has not been an automated process that could replace the current manual operation of plucking desirable embryos out of a development medium and placing them in an array suitable for further maturation treatments. The present invention is directed to providing a complete automated process of harvesting somatic embryos, which could replace the current manual operation.